(a) Field of the Invention
The invention relates to a photodynamic treatment for the preferential destruction of immunologically reactive cells without substantially affecting the normal cells or causing systemic toxicity for the patient.
(b) Description of Prior Art
Immunologic disorders are conditions or diseases that result from the production of immune cells recognizing normal cells and tissues as foreign. Cells with immunoreactivity towards normal cells or tissues induce damages in these normal cells and tissues either directly, through cellular effector mechanisms, or indirectly through antibodies, cytokines or other mediators. Such immunologic disorders are usually divided in alloimmune conditions and autoimmune conditions. Alloimmune disorders occur primarily in the context of allogeneic transplantation (bone marrow and other organs: kidney, heart, liver, lung, etc.). In the setting of bone marrow transplantation, donor immune cells present in the hematopoietic stem cell graft react towards host normal tissues, causing graft-versus-host disease (GVHD). The GVHD induces damage primarily to the liver, skin, intestine, lung, eyes and mouth. Autoimmune disorders are comprised of a number of arthritic conditions, such as rhumatoid arthritis, scleroderma and lupus erythematosus; endocrine conditions, such as diabetes mellitus; neurologic conditions, such as multiple sclerosis and myasthenia gravis; gastrointestinal conditions, such as Crohn's disease and ulcerative colitis; hematological disorders, such as autoimmune hemolytic anemia, etc. The immune reaction in both alloimmune and autoimmune disorders progresses to generate organ dysfunction and damage.
Despite important advances in treatment, immunologic complications remain the primary cause of failure of allogeneic transplantations, whether in hematopoietic stem cell transplantation (GVHD) or in solid organ transplantation (graft rejection). In addition, autoimmune disorders represent a major cause of both morbidity and mortality. Prevention and treatment of these immune disorders has relied mainly on the use of immunosuppressive agents, monoclonal antibody-based therapies, radiation therapy, and more recently molecular inhibitors. Significant improvement in outcome has occurred with the continued development of combined modalities, but for a small number of disorders and patients. However, for the most frequent types of transplantation (bone marrow, kidney, liver, heart and lung), and for most immune disorders (rhumatoid arthritis, connective tissue diseases, multiple sclerosis, etc.) resolution of the immunologic dysfunction and cure has not been achieved. Therefore, the development of new approaches for the prevention and treatment of immunologic disorders is critically needed particularly for those patients who are at high risk or whose disease has progressed and are refractory to standard immunosuppressive therapy. Allogeneic stem cell transplantation (AlloSCT) has been employed for the treatment of a number of malignant and non-malignant conditions. Allogeneic stem cell transplantation is based on the administration of high-dose chemotherapy with or without total body irradiation to eliminate malignant cells, and host hematopoietic cells. Normal hematopoietic donor stem cells are then infused into the patient in order to replace the host hematopoietic system. AlloSCT has been shown to induce increased response rates when compared with standard therapeutic options. One important issue that needs to be stressed when using AlloSCT relates to the risk of reinfusing immune cells that will subsequently recognize patient cells as foreign and cause GVHD. A variety of techniques have been developed that can deplete up to 99,999% of T cells from the stem cell graft. These techniques, including immunologic and physical purging, are not entirely satisfactory. One major consideration when purging stem cell grafts is to preserve the non-host-reactive T cells so that they can exert anti-infectious and anti-leukemia activity upon grafting. The potential of photodynamic therapy, in association with photosensitizing molecules capable of destroying immunologically reactive cells while sparing normal donor-non-reactive immune cells to purge hematopoeitic cell grafts in the preparation of AlloSCT or autologous stem cell transplantation (AutoSCT) and after AlloSCT in the context of donor lymphocyte infusions to eliminate recurring leukemia cells has largely been unexplored. To achieve eradication of T cells, several approaches have been proposed including:                1) in vitro exposure of the graft to monoclonal antibodies and immunotoxins against antigens present on the surface of T cells (anti-CD3, anti-CD6, anti-CD8, etc.);        2) in vitro selection by soybean agglutinin and sheep red blood cell resetting;        3) positive selection of CD34+ stem cells with or without additional negative selection of T cells;        4) in vivo therapy with combinations of anti-thymocyte globulin, or monoclonal antibodies,        5) in vivo or ex vivo treatment with photosensitizing agents; and        6) In vitro or ex vivo exposure of recipient-reactive donor T cells by monoclonal antibodies or immunotoxins targeting the interleukin 2 receptor or OX-40 antigen (Cavazzana-Calvo M. et al. (1990) Transplantation, 50:1-7; Tittle T. V. et al (1997) Blood 89:4652-58; Harris D. T. et al. (1999) Bone Marrow Transplantation 23:137-44).        
However, most of these methods are not specifically directed at the alloreactive T cell subset, but rather aiming at the elimination of either all T cells or broad T cell populations. This is associated with numerous problems, including disease recurrence, graft rejection, second malignancies and severe infections. In addition, the clinical relevance of several of these methods remains to be established.
There are many reports on the use of photodynamic therapy in the treatment of malignancies (Daniell M. D., Hill J. S. (1991) Aust. N. Z. J. Surg., 61: 340-348). One of these uses is described in U.S. Pat. Nos. 5,556,992 and 5,773,460, where novel photoactivable rhodamine derivatives are used for the photodynamic therapy of a cancer patient by destroying human cancer cells, wherein appropriate intracellular levels of the derivatives are achieved and irradiation with light of a suitable wavelength is applied. The method has been applied for cancers of various origins and for the eradication of viruses and pathogens (Raab O. (1990) Infusoria Z. Biol., 39: 524).
The initial experiments on the use of photodynamic therapy for cancer treatment using various naturally occurring or synthetically produced photoactivable substances were published early this century (Jesionek A., Tappeiner V. H. (1903) Muench Med Wochneshr, 47: 2042; Hausman W. (1911) Biochem. Z., 30: 276). In the 40's and 60's, a variety of tumor types were subjected to photodynamic therapy both in vitro and in vivo (Kessel, David (1990) Photodynamic Therapy of neoplastic disease, Vol. I, II, CRC Press. David Kessel, Ed. ISBN 0-8493-5816-7 (v. 1), ISBN 0-8493-5817-5 (v. 2)). Dougherty et al. and others, in the 70's and 80's, systematically explored the potential of oncologic application of photodynamic therapy (Dougherty T. J. (1974) J. Natl Cancer Inst., 51: 1333-1336; Dougherty T. J. et al. (1975) J. Natl Cancer Inst., 55: 115-121; Dougherty T. J. et al. (1978) Cancer Res., 38: 2628-2635; Dougherty T. J. (1984) Urol. Suppl., 23: 61; Dougherty T. J. (1987) Photochem. Photobiol., 45: 874-889). Several rhodamine derivatives were also found to display antitumor properties (U.S. Pat. Nos. 5,773,460 and 5,556,992). The specificity of these photosensitizing agents for malignant cells, which demonstrate high proliferation rates, prompted us to evaluate these agents for the elimination of immunologic cells.
Treatment of Immunologic Cells with Photodynamic Therapy
There is currently a lack of agents, which allow selective destruction of immunologic cells while leaving intact the normal non-pathogenic residual cellular population. Preferential uptake of photosensitive dye and cytotoxicity of photodynamic therapy against lymphoid cells (Greinix H. T., et al. Blood (1998) 92:3098-3104; Hunt D. W. et al (1999) Immunopharmacology, 41:31-44; Heykorenko E. A et al (1998) Immunopharmacology 40: 231-40); and macrophages (Heykorenko E. A. et al (1998) Immunopharmacology 40: 231-40; King D. E. et al 1999) Scand J. Immunol 49: 184-92) cells have been previously demonstrated and reviewed in Zic J. A. et al. Therapeutic Apheresis (1999) 3:50-62.
It would be highly desirable to be provided with photosensitizers, which possess the following characteristics:                i) preferential localization outside the nucleus and uptake by the immunologic cells;        ii) upon application of appropriate light intensities, killing those cells which have accumulated and retained the photosensiting agents;        iii) sparing a sufficient proportion of the normal hematopoietic stem cell compartment from the destructive effects of activated photosensitizers; and        iv) potential utilization of photosensitizers for hematopoietic stem cell purging of immunologic cells in preparation for allogeneic or autologous stem cell transplantation.        v) Potential utilization of photosensitizers for ex vivo elimination of cells of the immune system in patients with immunological disorders.The Rhodamine Dyes        
Rhodamine 123 (2-(6-amino-3-imino-3H-xanthen-9-yl)benzoic acid methyl ester)hydrochloride, a lipophilic cationic dye of the pyrylium class which can disrupt cellular homeostasis and be cytostatic or cytotoxic upon high concentration exposure and/or photodynamic therapy, although with a very poor quantum yield (Darzynkiewicz Z., Carter S. (1988) Cancer Res., 48: 1295-1299). It has been used in vitro as a specific fluorescent stain for living mitochondria. It is taken up and is preferentially retained by many tumor cell types, impairing their proliferation and survival by altering membrane and mitochondrial function (Oseroff A. R. (1992) In Photodynamic therapy (Henderson B. W., Dougherty T. J., eds) New York: Marcel Dekker, pp. 79-91). In vivo, chemotherapy with rhodamine 123 can prolong the survival of cancerous mice, but, despite initial attemps to utilize rhodamine 123 in the treatment of tumors, the systemic toxicity may limit its usefulness (Bernal, S. D., et al. (1983) Science, 222: 169; Powers, S. K. et al. (1987) J. Neurosur., 67: 889).
U.S. Pat. No. 4,612,007 issued on Sep. 16, 1986 in the name of Richard L. Edelson, discloses a method for externally treating human blood, with the objective of reducing the functioning lymphocyte population in the blood system of a human subject. The blood, withdrawn from the subject, is passed through an ultraviolet radiation field in the presence of a dissolved photoactive agent capable of forming photoadducts with lymphocytic-DNA. This method presents the following disadvantages and deficiencies. The procedure described is based on the utilization of known commercially available photoactive chemical agents for externally treating patient's blood, leaving immune cells from other sites intact in the process. According to Richard L. Edelson, the method only reduces, does not eradicate, the target cell population. This treatment strategy does not incorporate any attempt to enhance the immunoreactivity of target cells. Moreover, the wavelength range of UV radiation used in the process proposed by Richard L. Edelson could be damageable to the normal cells.
International Application published on Jan. 7, 1993 under International publication number WO 93/00005, discloses a method for inactivating pathogens in a body fluid while minimizing the adverse effects caused by the photosensitive agents. This method essentially consists of treating the cells in the presence of a photoactive agent under conditions that effect the destruction of the pathogen, and of preventing the treated cells from contacting additional extracellular protein for a predetermined period of time. This method concerned the eradication of infectious agents from collected blood and its components, prior to storage or transfusion, and does not impede on the present invention.
It would be highly desirable to be provided with a new use of rhodamine derivatives in the treatment of immunologic cells, which overcomes these drawbacks while having no substantial systemic toxicity for the patient.